Type I diabetes is an autoimmune disease mediated by CD4 T cells that leads to the destruction of the insulin producing beta cells in the pancreatic islets of Langerhans. Once the beta cells are destroyed, blood sugar levels are unable to be controlled due to the lack of insulin, which leads to hyperglycemia. The only treatment for type I diabetes is life-long insulin therapy, which is costly and requires constantly measuring blood glucose levels. Therefore it would be nice to develop a therapy that specifically targets the perpetrators to halt the destruction of the beta cells in newly diagnosed patients or in combination with islet cell transplants. We feel that antigen-specific regulatory T cells (Tregs) would be able to accomplish our goal of selective inhibiting the autoreactive T cells without compromising the immune system. We propose a protocol for the rapid, selective expansion of antigen-specific Tregs using a variant peptide. Unlike altered peptide ligands (APLs) which have substitutions at amino acids that contact the T cell receptor, we propose that by changing amino acids that contact the major histocompatibility complex (MHC) we can alter T cell fate without the disadvantages of APLs. Clinical trials in humans had to be halted due to exacerbation of disease, even though APLs showed promise in animal models. The exacerbation was due to the outgrowth of a population of T cells that were specific for the APLs. We would like to harness the advantages of APLs without the disadvantages through the use of our MHC variant peptides, which have substitions at MHC contact positions. By altering the stability of the peptide:MHC complex we hypothesize that we can control T cell activation, thereby allowing us to selectively expand a regulatory population. Our first aim is to determine the stability of the peptide:MHC complex and measure the suppressive capacity of the expanded Tregs. We plan on using binding exchange reactions to measure the stability of the complex. To measure the suppressive capacity, we will determine the amount of suppressive cytokines being produced, and employ in vitro and in vivo models of suppression. The in vivo protection from development of spontaneous or inducible diabetes assays will show the usefulness of this proposal to the development of possible therapeutics for human use. The second aim will be to determine the signaling cascades involved in the expansion of Tregs since we believe that the unstable peptide:MHC complex leads to differential signaling within the cells. We will use both flow cytometry methods and western blotting for the detection of the phosphorylated forms of key signaling molecules in kinetic studies of activation. The significance of our proposal is that MHC variant peptides can be utilized to selectively expand antigen specific Tregs which can then be used to prevent diabetes shortly after diagnosis when beta cell destruction is incomplete, and to downregulate the self-reactive cells prior to an islet cell transplant, which would better ensure a successful long-term survival of the graft.